Project Summary/Abstract The main goal of the lab is to provide detailed structural and functional insight into serotonin signaling and transport, and elucidate how drugs and medications modulate receptor and transporter function. Serotonin (5- hydroxytryptamine, 5-HT) regulates much of human physiology in- and outside the CNS, including cognition, mood, endocrine function and cardiovascular development. However, despite 5-HT's physiological and medical importance, atomic-level insight into the mechanisms of 5-HT signaling and transport, and how drugs interact with these molecular targets, has remained largely elusive. This lack of understanding has led to drug safety issues in the past, and greatly hindered the exploration of many 5-HT targets for novel therapeutic applications. Our lab is thus focused on two grossly understudied 5-HT targets. The first major direction of the lab is to elucidate ligand binding and activation of the 5-HT1E receptor, the least understood 5-HT receptor whose physiological role is unknown. Unlike for all other 5-HT receptors, there are no selective compounds for 5- HT1ER, which raises important questions about the molecular architecture of its binding pocket and mechanisms of receptor activation. To answer these questions, we will determine the cryoEM structure of G protein-coupled 5-HT1ER, which will provide detailed structural insight into the ligand binding pocket architecture, and the receptor's conformational changes responsible for G protein activation. The second major project is focused on the vesicular monoamine transporter 2 (VMAT2), an essential transporter for storage and regulation of neurotransmitter signaling, and target for a variety of disorders from diabetes to amphetamine abuse. Despite its biological and medical significance, there is a lack of atomic-level insight into where and how substrates, drugs, or medications bind, or how they modulate VMAT2 function. We will therefore determine crystal structures of VMAT2 bound to various substrates and drugs, elucidate fundamental mechanisms of transport and drug modulation, and characterize the precise architecture of different binding sites. We will further use our molecular insights in structure-based drug discovery, to develop pharmacologically and chemically novel tool compounds for future studies of VMAT2 (patho)biology in in vitro and in vivo. Our studies are both innovative technically and conceptually, as the unique combination of structural, pharmacological, and drug discovery approaches will provide an unprecedented view into the molecular mechanisms of two neglected and understudied 5-HT drug targets.